Label-free quantitative proteomic analysis of milk fat globule membrane proteins in porcine colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are nutritional components with various biological functions. This study aimed to analyze and compare MFGM proteins in porcine colostrum (PC) and porcine mature milk (PM), via label-free quantitative proteomics. In total, 3917 and 3966 MFGM proteins were identified in PC and PM milk, respectively. A total of 3807 common MFGM proteins were found in both groups, including 303 significant differentially expressed MFGM proteins. Gene Ontology (GO) analysis revealed that the differentially expressed MFGM proteins were mainly related to the cellular process, cell, and binding. The dominant pathway of the differentially expressed MFGM proteins was related to the phagosome according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. These results reveal crucial insights into the functional diversity of MFGM proteins in porcine milk during lactation and provide theoretical guidance for the development of MFGM proteins in the future.

Protocol for high-throughput compound screening using flow cytometry in THP-1 cells.

Flow cytometry is a valuable method for analyzing protein expressions at the single cell level but can be difficult to apply to large numbers of samples. This protocol provides instructions to perform a high-throughput small molecule screen using flow cytometry analysis of THP-1 cells, a human monocytic leukemia cell line. We describe a methodology for identifying compounds that regulate PD-L1 surface expression in IFN-γ-stimulated cells, which has been successfully used to screen a collection of ∼200,000 compounds. For complete details on the use and execution of this protocol, please refer to Zavareh et al. (2020).

Siglec-15 as an Emerging Target for Next-generation Cancer Immunotherapy.

Immunomodulatory agents blocking the PD-1/PD-L1 pathway have shown a new way to treat cancer. The explanation underlying the success of these agents may be the selective expression of PD-L1 with dominant immune-suppressive activities in the tumor microenvironment (TME), supporting a more favorable tumor response-to-toxicity ratio. However, despite the big success of these drugs, most patients with cancer show primary or acquired resistance, calling for the identification of new immune modulators in the TME. Using a genome-scale T-cell activity array in combination with bioinformatic analysis of human cancer databases, we identified Siglec-15 as a critical immune suppressor with broad upregulation on various cancer types and a potential target for cancer immunotherapy. Siglec-15 has unique molecular features compared with many other known checkpoint inhibitory ligands. It shows prominent expression on macrophages and cancer cells and a mutually exclusive expression with PD-L1, suggesting that it may be a critical immune evasion mechanism in PD-L1-negative patients. Interestingly, Siglec-15 has also been identified as a key regulator for osteoclast differentiation and may have potential implications in bone disorders not limited to osteoporosis. Here, we provide an overview of Siglec-15 biology, its role in cancer immune regulation, the preliminary and encouraging clinical data related to the first-in-class Siglec-15 targeting mAb, as well as many unsolved questions in this pathway. As a new player in the cancer immunotherapeutic arena, Siglec-15 may represent a novel class of immune inhibitors with tumor-associated expression and divergent mechanisms of action to PD-L1, with potential implications in anti-PD-1/PD-L1-resistant patients.

Static magnetic fields accelerate osteogenesis by regulating FLRT/BMP pathway.

OBJECTIVES: Static magnetic fields (SMF) have been proved to enhance osteogenic differentiation in mesenchymal stem cells (MSCs). However, the effect of SMF on mandibular condylar chondrocytes (MCCs) are less investigated, which contributes to the vertical formation of mandible. The purpose of the present study was to identify whether SMF accelerate the osteogenesis on mature condylar cartilage and explore the potential regulatory mechanism. METHODS: In this study, we presented a 280 mT SMF stimulation set-up to investigate the genomic effects of SMF exposure on MCCs differentiation and osteoblast-related factor secretion in vitro. Induced by Oricell™ for osteogenesis, MCCs from primary SD Rat were stimulated with or without SMF for cell culture. Cell proliferation was determined by CCK-8. The enhanced osteogenetic capacity of the SMF stimulated MCCs was identified by Alizarin red staining (ARS). Additionally, the effects of SMF on the expression of transmembrane protein marker (FLRT3), terminal differentiation markers (BMP2), and transcription factors (Smad1/5/8) were quantified by Western blot and immunofluorescence analysis. RESULTS: Compared with the control group, SMF decreased the proliferation of MCCs (p < 0.05) after 14 days osteogenesis-specific induction. The mineral synthesis of MCCs was upregulated by SMF (p < 0.0001). The expression of BMP2, Smad1/5/8 showed decrease trends while the protein level of FLRT3 acted in contrary manner (p < 0.05). CONCLUSIONS: Our findings emphasized the ability of osteogenesis positively respond to SMF stimulation by exhibiting enhanced differentiation via FLRT/BMP signaling.

The Effect of Shu Xue Tong Treatment on Random Skin Flap Survival via the VEGF-Notch/Dll4 Signaling Pathway.

Objective: To determine the effect of Shu Xue Tong treatment on random skin flap survival. Methods: Twenty-four male Sprague-Dawley (SD) rats were administered Shu Xue Tong or normal saline. On postoperative day 7, flap survival area, flap angiogenesis angiography, blood flow and vascular endothelial growth factor (VEGF), Notch1, and Delta-like ligand 4 (Dll4) expression were assessed. Results: The mean flap survival area, blood vessel regeneration, microcirculatory flow, and expression of VEGF were enhanced with Shu Xue Tong treatment relative to the control. However, in Shu Xue Tong-treated rat flap, Notch1 and Dll4 levels were significantly reduced. Conclusions: These results demonstrate the beneficial action of Shu Xue Tong on random skin flap survival and suggest that its mechanism of action involves the promotion of angiogenesis via the VEGF-Notch1/Dll4 signaling pathway.

Effectiveness of different proteases in reducing allergen content and IgE-binding of raw peanuts.

The effectiveness of some non-specific proteases in reducing raw peanut allergenicity was investigated. Peanut kernels were treated by Alcalase, papain, Neutrase and bromelain, respectively. The residues of major peanut allergens Ara h 1, Ara h 2 and Ara h 6 were determined by sandwich ELISA and SDS-PAGE, and the allergenicities of treated peanuts were compared to that of untreated peanuts by western blot. All tested proteases were effective in reducing Ara h 1, but their effectiveness in hydrolyzing Ara h 2 and Ara h 6 varied greatly. The maximal reductions of extractable Ara h 1, Ara h 2 and Ara h 6 were 100%, 100% and 99.8%, respectively, achieved by Alcalase hydrolysis. Alcalase was more effective in overall allergenicity reduction; bromelain and Neutrase were the least effective in reducing Ara h 2 and Ara h 6, respectively. The hydrolysis of original allergens also produced some smaller peptides with strong IgE-binding.

Proposal of a study protocol of a preliminary double-blind randomized controlled trial. Verifying effects of selenium supplementation on selenoprotein p and s genes expression in protein and mRNA levels in subjects with coronary artery disease: selenegene.

BACKGROUND: Selenium is the component of selenocystein amino acid, which itself is the building block of selenoproteins having diverse effects on various aspects of the human health. Among these proteins, selenoprotein P is the central to the distribution and homeostasis of selenium, and selenoprotein S as a transmembrane protein is associated with a range of inflammatory markers, particularly in the context of cardiovascular disease. It is known that selenium status outside of the normal range is considered to confer different benefits or adverse cardiovascular risk factors. Therefore, for the first time, we aimed to verify effects of Selenium supplementation on Selenoprotein P and S Genes Expression in Protein and mRNA Levels in Subjects with Coronary Artery Disease (CAD). METHODS: This is the study protocol of a double blinded randomized clinical trial on 130 subjects with angiographically documented stenosis of more than 75% in one or more coronary artery vessels. In this 60-day study, 65 patients in each group received either a 200mg selenium yeast or placebo tablets once daily. During the study, subjects were followed by phone calls and visited our clinic twice to repeat baseline measurements. We hypothesized that our finding would enable a more basic and confirmed understanding for the effect of selenium supplementation by investigating its effect on gene expression levels in people with CAD. DISCUSSION: Upon confirmation of this hypothesis, the beneficial effect of inflammation regulation by supplementation with micronutrients could be considered for subjects with CVD.

Comprehensive characterization of bioactive peptides from Buffalo (Bubalus bubalis) colostrum and milk fat globule membrane proteins.

Milk fat is dispersed in milk as small spherical globules stabilized in the form of emulsion by its surrounding membrane, often referred to as fat globule membrane (FGM). Buffalo, a major milking mammal of Asia and second most milking mammal across the globe presents physicochemical features different from that of other ruminant species containing higher content of lipids and proteins. The present study describes characterization of FGM proteins isolated from both buffalo milk and colostrum. A detailed proteomic analysis of peptides generated by in vitro gastrointestinal simulation digestion of buffalo milk and colostrum FGM fractions was performed by nLC-ESI MS/MS. The peptide based clustering of FGM proteins unravelled association of membrane proteins in fat transport, enzymatic activity, general transport, defence, cell signalling, membrane/protein trafficking protein synthesis/binding/folding including unknown functions. Gene annotation, STRING and YLoc analyses provided putative insights into major secretory pathways in milk and colostrum FGM peptides, interactive protein networks including their sub cellular localization. The peptides of milk and colostrum FGM offered cellular protection as powerful antioxidants indicated their promising perspectives in commercial formulations and nutraceuticals.

NT1721, a novel epidithiodiketopiperazine, exhibits potent in vitro and in vivo efficacy against acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by heterogeneous genetic and epigenetic changes in hematopoietic progenitors that lead to abnormal self-renewal and proliferation. Despite high initial remission rates, prognosis remains poor for most AML patients, especially for those harboring internal tandem duplication (ITD) mutations in the fms-related tyrosine kinase-3 (FLT3). Here, we report that a novel epidithiodiketopiperazine, NT1721, potently decreased the cell viability of FLT3-ITD+ AML cell lines, displaying IC50 values in the low nanomolar range, while leaving normal CD34+ bone marrow cells largely unaffected. The IC50 values for NT1721 were significantly lower than those for clinically used AML drugs (i.e. cytarabine, sorafenib) in all tested AML cell lines regardless of their FLT3 mutation status. Moreover, combinations of NT1721 with sorafenib or cytarabine showed better antileukemic effects than the single agents in vitro. Combining cytarabine with NT1721 also attenuated the cytarabine-induced FLT3 ligand surge that has been linked to resistance to tyrosine kinase inhibitors. Mechanistically, NT1721 depleted DNA methyltransferase 1 (DNMT1) protein levels, leading to the re-expression of silenced tumor suppressor genes and apoptosis induction. NT1721 concomitantly decreased the expression of EZH2 and BMI1, two genes that are associated with the maintenance of leukemic stem/progenitor cells. In a systemic FLT3-ITD+ AML mouse model, treatment with NT1721 reduced tumor burdens by > 95% compared to the control and significantly increased survival times. Taken together, our results suggest that NT1721 may represent a promising novel agent for the treatment of AML.

Fluorescent Detection of Lipid Droplets and Associated Proteins.

Excess lipid is stored in intracellular organelles known as lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets in fixed or live cells with BODIPY 493/503 are included. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet-associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology. Immunofluorescent detection of perilipin 2, a broadly expressed, lipid droplet-associated protein, widely used as a marker for lipid droplet accumulation, is presented as an example. Finally, a simple protocol for enhancing lipid droplet accumulation through supplementation with excess fatty acid is included. © 2016 by John Wiley & Sons, Inc.

Comparative proteomics of milk fat globule membrane in goat colostrum and mature milk.

As an important nutrient source in large area of world, the composition and nutritional value of goat milk are not well deliberated. Detailed annotation of protein composition is essential to address the physiological and nutritional value of goat milk. In the present study, 423 colostrum and mature goat milk fat globule membrane (MFGM) proteins were identified. The abundance of 189 proteins was significantly different between colostrums and mature milk MFGM. The acute phase proteins were higher in colostrums MFGM than those in mature milk MFGM which protected newborns at the beginning of life. Proteins related to synthesis and secretion were conserved through lactation to ensure the milk production. Of note, long term depression (LTD) proteins were observed in colostrum and mature milk MFGM. Milk LTD proteins could be potential biomarkers for diagnosis of lactation related depressive syndromes and should be taken into considerations of their effects on newborns.

Fully automated multidimensional reversed-phase liquid chromatography with tandem anion/cation exchange columns for simultaneous global endogenous tyrosine nitration detection, integral membrane protein characterization, and quantitative proteomics mapping in cerebral infarcts.

Protein tyrosine nitration (PTN) is a signature hallmark of radical-induced nitrative stress in a wide range of pathophysiological conditions, with naturally occurring abundances at substoichiometric levels. In this present study, a fully automated four-dimensional platform, consisting of high-/low-pH reversed-phase dimensions with two additional complementary, strong anion (SAX) and cation exchange (SCX), chromatographic separation stages inserted in tandem, was implemented for the simultaneous mapping of endogenous nitrated tyrosine-containing peptides within the global proteomic context of a Macaca fascicularis cerebral ischemic stroke model. This integrated RP-SA(C)X-RP platform was initially benchmarked through proteomic analyses of Saccharomyces cerevisiae, revealing extended proteome and protein coverage. A total of 27 144 unique peptides from 3684 nonredundant proteins [1% global false discovery rate (FDR)] were identified from M. fascicularis cerebral cortex tissue. The inclusion of the S(A/C)X columns contributed to the increased detection of acidic, hydrophilic, and hydrophobic peptide populations; these separation features enabled the concomitant identification of 127 endogenous nitrated peptides and 137 transmembrane domain-containing peptides corresponding to integral membrane proteins, without the need for specific targeted enrichment strategies. The enhanced diversity of the peptide inventory obtained from the RP-SA(C)X-RP platform also improved analytical confidence in isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses.

Extraction and identification of membrane proteins from black widow spider eggs.

The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider (Latrodectus tredecimguttatus), and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCl buffer containing 4% CHAPS and 2% DTT (pH 7.4). SDS-PAGE combined with nLC-MS/MS identified 39 proteins with membrane-localization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development.

Identification of extracellularly phosphorylated membrane proteins.

Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling.

Protective effects of ginsenoside Rg1 on aging Sca-1⁺ hematopoietic cells.

In adults, bone hematopoietic cells are responsible for the lifelong production of all blood cells. It is affected in aging, with progressive loss of physiological integrity leading to impaired function by cellular intrinsic and extrinsic factors. However, intervention measures, which directly inhibit the aging of hematopoietic cells, remain to be investigated. In the present study, 10 µmol/l ginsenoside Rg1 (Rg1) markedly alleviated the aging phenotypes of Sca­1+ hematopoietic cells following in vitro exposure. In addition, the protective effects of ginsenoside Rg1 on the aging of Sca­1+ hematopoietic cells was confirmed using a serial transplantation assay in C57BL/6 mice. The mechanistic investigations revealed that Rg1­mediated Sca­1+ hematopoietic cell aging alleviation was linked to a series of characteristic events, including telomere end attrition compensation, telomerase activity reconstitution and the activation of genes involved in p16­Rb signaling pathways. Based on the above results, it was concluded that ginsenoside Rg1 is a potent agent, which acts on hematopoietic cells to protect them from aging, which has implications for therapeutic approaches in hemopoietic diseases.

Antioxidant Opuntia ficus-indica Extract Activates AHR-NRF2 Signaling and Upregulates Filaggrin and Loricrin Expression in Human Keratinocytes.

Opuntia ficus-indica (OFI) is a cactus species widely used as an anti-inflammatory, antilipidemic, and hypoglycemic agent. It has been shown that OFI extract (OFIE) inhibits oxidative stress in animal models of diabetes and hepatic disease; however, its antioxidant mechanism remains largely unknown. In this study, we demonstrated that OFIE exhibited potent antioxidant activity through the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and the downstream antioxidant enzyme NAD(P)H: quinone oxidoreductase 1 (NQO1), which inhibited the generation of reactive oxygen species in keratinocytes challenged with tumor necrosis factor α or benzo[α]pyrene. The antioxidant capacity of OFIE was canceled in NRF2 knockdown keratinocytes. OFIE exerted this NRF2-NQO1 upregulation through activation of the aryl hydrocarbon receptor (AHR). Moreover, the ligation of AHR by OFIE upregulated the expression of epidermal barrier proteins: filaggrin and loricrin. OFIE also prevented TH2 cytokine-mediated downregulation of filaggrin and loricrin expression in an AHR-dependent manner because it was canceled in AHR knockdown keratinocytes. Antioxidant OFIE is a potent activator of AHR-NRF2-NQO1 signaling and may be beneficial in treating barrier-disrupted skin disorders.

[Effect of SIRT6/NF-κB signal axis in delaying hematopoietic stem/progenitor cell senescence with ginsenoside Rg1].

OBJECTIVE: To investigate the effect of SIRT6/NF-κB signal axis in delaying hematopoietic stem/progenitor cell senescence with ginsenoside Rg1, in order to provide theatrical and experimental basis for looking for methods for delaying HSC senescence. METHOD: Sca-1 + HSC/HPC was isolated by magnetic cell sorting (MACS) and divided into five groups: the normal control group, the aging group, the positive control group, the Rg1 anti-senescence group, and the Rg1-treated group. Senescence-associated ß-galactosidase (SA-ß-Gal) staining, cell cycle analysis and hemopoietic progenitor cell mix (CFU-Mix) were adopted to determine the effect Rg1 in delaying or treating Sca-1 + HSC/HPC senescence biology. The mRNA and protein of senescence regulation molecules SIRT6 and NF-KB were examined by realtime fluorescence quantitative PCR (FQ-PCR) and western blotting. RESULT: Compared with the senescence group, the Rg1 anti-senescence group and the Rg1-treated group showed lower percentage in SA-ß-Gal-stained positive cells, decreased cell proportion in G1 phase, increased number of CFU-Mix, up-regulated in SIRT6 mRNA and protein expression, down-regulation in NF-KB mRNA and protein expression. The Rg1 anti-senescence group showed more evident changes in indexes than the Rg1-treated group. CONCLUSION: Rg, may inhibit Sca-1 + HSC/HPC senescence induced by t-BHP by regulating SIRT6/NF-KB signal path.

Protective role of dietary-supplemented selenium and vitamin E in heat-induced apoptosis and oxidative stress in mice testes.

This study evaluated the role of selenium (0.5 ppm selenium/kg diet) and vitamin E (200 mg alpha-tocopherol/kg diet) on spermatogenesis after scrotal hyperthermia (42 °C, 30 min) in six different groups of male Balb/c mice; Control, Heat shock, Selenium, Selenium+heat shock, Vitamin E and Vitamin E+heat shock. Markers of the stress responses, hypoxia and oxidative stress, were evaluated in testis after the hyperthermic shock. Hyperthermia caused an elevated mRNA expression of hypoxia-inducible factor-1 alpha, haem oxygenase-1 (HMOX-1) and also glutathione peroxidase activity and reactive oxygen species (ROS). Apoptosis was evaluated by TUNEL assay and further by mRNA expression of Bcl-2, caspase 3, 8, 9, bid and AKT. TUNEL assay showed significant increase in apoptotic index of spermatogenic cells, whereas decrease in mRNA expression of Bcl-2, AKT and increase in caspase 3, 8, 9 and Bid in heat-shock group were observed. A significant decrease in sperm motility was also seen in heat-shock group in comparison with control group. These observations clearly indicate the development of oxidative stress and apoptosis after hyperthermia. Further analysis in Selenium+heat shock and Vitamin E+heat shock groups showed protective behaviour as compared to effects in heat-shock group which could be of therapeutic interest in future studies.

Parenteral nutrition-associated liver injury and increased GRP94 expression prevented by ω-3 fish oil-based lipid emulsion supplementation.

OBJECTIVE: Parenteral nutrition in infants with gastrointestinal disorders can be lifesaving, but it is also associated with parenteral nutrition-associated liver disease. We investigated the effects of incorporating ω-3 fish oil in a parenteral nutrition mixture on signs of parenteral nutrition-associated liver disease and explored the mechanism involved in this process. METHODS: Seven-day-old New Zealand rabbits were divided into 3 groups of 8, and for 1 week they were infused via the right jugular vein with standard total parenteral nutrition with soybean oil (TPN-soy) or TPN with ω-3 fish oil-based lipid emulsion (TPN-FO), or naturally nursed with rabbit milk (control). Serum and liver tissues were analyzed for serological indicators and pathology, respectively. Reverse-transcriptase polymerase chain reaction was used to evaluate the messenger RNA levels of the endoplasmic reticulum stress chaperone protein glucose-regulated protein 94 (GRP94) in liver tissues and GRP94 protein levels were compared through immunohistochemistry and Western blot assays. RESULTS: TPN-soy animals had significantly higher serum total bilirubin, direct bilirubin, and γ-glutamyl transpeptidase and lower serum albumin than the controls (P < 0.01, each) or the TPN-FO group, which were similar to the controls (P < 0.01 cf. TPN). Damage to liver tissues of the TPN-FO group was much less than that of the TPN-soy group. GRP94 messenger RNA and protein levels in liver tissues of TPN-soy animals were significantly higher than that of the controls or TPN-FO rabbits, which were similar to the controls. CONCLUSIONS: Incorporating ω-3 fish oil in parenteral nutrition emulsion greatly prevented liver dysfunction and liver tissue damage in week-old rabbit kits, possibly by preventing endoplasmic reticulum stress.

Membrane carbonic anhydrase IX expression and relapse risk in resected stage I-II non-small-cell lung cancer.

BACKGROUND: Adjuvant chemotherapy reduces recurrences of non-small-cell lung cancer (NSCLC). To determine which patients need adjuvant chemotherapy, we assessed factors associated with time to relapse (TTR). METHODS: In 230 resected stage I-II NSCLCs, we correlated immunohistochemistry scores for factors associated with cell growth rate, growth regulation, hypoxia, cell survival, and cell death with TTR. RESULTS: With a median follow-up of 82 months (1-158) for those alive and relapse free at last follow-up, median time to recurrence was not reached. The 2- and 5-year probabilities of maintaining freedom from recurrence were 80.7% (95% confidence interval, 75.3%, 86.4%) and 74.6% (95% confidence interval, 68.6%, 81.2%), respectively. TTR curves flattened at an apparent cure rate of 70%. In multicovariate Cox models, factors correlating with shorter TTR were membranous carbonic anhydrase IX (mCAIX) staining (any versus none, hazard ratio = 2.083, p = 0.023) and node stage (N1 versus N0, hazard ratio = 2.591, p = 0.002). mCAIX scores correlated positively with tumor size, grade, squamous histology, necrosis, mitoses, Ki67, p53, nuclear DNA methyltransferase 1, and cytoplasmic enhancer-of-split-and-hairy-related protein, and they correlated inversely with papillary histology, epidermal growth factor receptor mutation (trend), copper transporter-1, and cytoplasmic hypoxia-inducible factor-1α, vascular endothelial growth factor, DNA methyltransferase 1, and excision repair cross-complementing rodent repair deficiency, complementation group 1. CONCLUSION: Nodal stage and mCAIX immunohistochemistry were the strongest independent predictors of shorter TTR in resected NSCLCs. mCAIX correlated with tumor size, markers of tumor proliferation and necrosis, and tumor genetic characteristics, and it paradoxically correlated inversely with the hypoxia markers, hypoxia-inducible factor-1α and vascular endothelial growth factor. Presence of mCAIX could help determine patients with high risk of recurrence who might require adjuvant chemotherapy.